Rice WRKY gene and application thereof in regulating anthocyanin content of crops

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to a rice WRKY gene and application thereof in regulating anthocyanin content of crops. The application provides a rice WRKY gene and application thereof in regulating anthocyanin content of crops, and the rice WRKY gene comprises OsWRKY1 gene and / or OsWRKY57 gene; the OsWRKY1 amino acid sequence encoded by the gene is shown as SEQ ID NO:6; the OsWRKY57 amino acid sequence encoded by the gene is shown as SEQ ID NO:18. The application proves that OsWRKY1 is a positive regulation factor in anthocyanin synthesis of rice, OsWRKY57 is a negative regulation factor in anthocyanin synthesis of rice, and the application provides an effective way for breeding black rice.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to rice. WRKY Genes and their application in regulating crop anthocyanin content. Background Technology

[0002] Black rice is a type of colored rice, named for the black or dark brown anthocyanins enriched in its bran. It's a special rice variety rich in natural pigments, and its rice quality surpasses that of traditional rice varieties, particularly in its unique advantages in chemical composition, physical properties, and aroma. The anthocyanins in its pericarp possess strong antioxidant properties, effectively scavenging reactive oxygen species (ROS) such as hydroxyl radicals and DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radicals. These significant nutritional and functional properties have led to increasing research interest in black rice, making it a crucial raw material for developing functional foods.

[0003] Anthocyanins and proanthocyanidins are widely distributed secondary metabolites in plants with various biological functions. They are also two very important natural pigments in colored rice, both belonging to the flavonoid family. Their biosynthesis shares most of the synthases and has similar regulatory patterns. The flavonoid metabolic pathway is one of the most studied metabolic pathways. Transcription factors can bind to cis-elements on the promoters of structural genes in the metabolic pathway, thereby activating or inhibiting the transcriptional level of structural genes to regulate metabolite synthesis. Many regulatory proteins control the spatiotemporal expression of genes involved in anthocyanin synthesis exist in the anthocyanin synthesis pathway. MYB, bHLH, and WD40 repeat proteins are the main transcription factors regulating flavonoid synthesis. MYB-type transcription factors can regulate anthocyanin synthesis genes independently, or they can form the MBW (MYB-bHLH-WDR) complex with bHLH and WDR to activate the expression of structural genes.

[0004] However, current research on anthocyanin regulation mechanisms focuses on the MBW complex, while the regulatory mechanisms of other types of transcription factors in anthocyanin biosynthesis remain relatively scarce. Therefore, exploring new candidate genes from the anthocyanin synthesis regulatory network, conducting in-depth research on the molecular mechanisms of rice anthocyanin biosynthesis, and applying these findings to black rice breeding has become an urgent problem to be solved in this field. Summary of the Invention

[0005] To address the lack of genes in existing technologies that can regulate anthocyanin synthesis in rice, this invention provides a rice... WRKY Genes and their application in regulating crop anthocyanin content, specifically including the following technical solutions: This invention provides rice WRKY The application of genes in regulating anthocyanin content in crops, specifically in rice. WRKYGenes include OsWRKY1 Genes and / or OsWRKY57 Gene; The OsWRKY1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:6; The OsWRKY57 The amino acid sequence encoded by the gene is shown in SEQ ID NO:18.

[0006] Preferably, the crop includes monocotyledonous plants.

[0007] Preferably, the regulation of crop anthocyanin content includes any one or more of the following aspects: 1) Overexpression OsWRKY1 Genes increase anthocyanin content in crops; 2) Knockout or suppression OsWRKY1 Gene expression reduces anthocyanin content in crops; 3) Knockout or suppression OsWRKY57 Gene expression increases anthocyanin content in crops; 4) Overexpression OsWRKY57 Gene expression reduces anthocyanin content in crops.

[0008] Preferably, the regulation of crop anthocyanin content includes regulating the anthocyanin content in crop grains.

[0009] This invention also provides a method for regulating rice WRKY Application of gene-expressing biomaterials in increasing anthocyanin content in crops, specifically rice. WRKY Genes include OsWRKY1 Genes and / or OsWRKY57 Gene; The OsWRKY1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:6; The OsWRKY57 The amino acid sequence encoded by the gene is shown in SEQ ID NO:18.

[0010] This invention also provides a method for regulating rice WRKY Gene-expressing biological materials, wherein the biological materials include any one or more of the following aspects: 1) Overexpression OsWRKY1 Primer pairs for genes; 2) Contains the above OsWRKY1 Gene recombination vectors; 3) Contains the above OsWRKY1 Recombinant microorganisms of the gene or the recombinant vector described in (2); 4) Knock out the above OsWRKY57 The target sequence of the gene; 5) Targeting the above OsWRKY57 Primer pairs for the target sequence of a gene; 6) Overexpression OsWRKY57 Primer pairs for genes; 7) Contains the above OsWRKY57 Gene recombination vectors; 8) Contains the above OsWRKY57 Recombinant microorganisms of the gene or the recombinant vector described in 7); The OsWRKY1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:6; The OsWRKY57 The amino acid sequence encoded by the gene is shown in SEQ ID NO:18.

[0011] Preferably, the nucleotide sequences of the primer pair described in 1) are as shown in SEQ ID NO:3 and SEQ ID NO:4; Preferably, 2) the recombinant vector includes an overexpression recombinant vector; the recombinant vector includes a backbone vector, and the backbone vector includes a plasmid vector; Preferably, 3) the recombinant microorganism includes an overexpressed recombinant microorganism, the recombinant microorganism includes a basic microorganism, and the basic microorganism includes bacteria.

[0012] Preferably, the target sequence described in 4) is as shown in SEQ ID NO:19; Preferably, the primer pair for the target sequence in 5) is as shown in SEQ ID NO:20 and SEQ ID NO:21; Preferably, the primer pair described in 6) is as shown in SEQ ID NO:22 and SEQ ID NO:23; Preferably, 7) the recombinant vector includes a knockout recombinant vector; the recombinant vector includes a backbone vector, and the backbone vector includes a plasmid vector; Preferably, 8) the recombinant microorganism includes a knockout recombinant microorganism; the recombinant microorganism includes a basic microorganism, and the basic microorganism includes bacteria.

[0013] This invention also provides a breeding method for increasing the anthocyanin content in crop grains, comprising any one or two of the following steps: 1) Overexpression of the above in crops OsWRKY1 Genes to increase the anthocyanin content in crop grains; The OsWRKY1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:6; 2) Knockout or inhibition in crops OsWRKY57Gene expression to increase anthocyanin content in crop grains; The OsWRKY57 The amino acid sequence encoded by the gene is shown in SEQ ID NO:18.

[0014] This invention also provides a method for creating a biological model with low anthocyanin content, comprising any one or two of the following steps: 1) Overexpression of the above in crops OsWRKY57 Genes to reduce anthocyanin content in crops; The OsWRKY57 The amino acid sequence encoded by the gene is shown in SEQ ID NO:18; 2) Knockout or inhibition in crops OsWRKY1 Gene expression to reduce anthocyanin content in crops; The OsWRKY1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:6.

[0015] The beneficial effects of this invention are as follows: This invention provides rice WRKY The application of genes in regulating anthocyanin content in crops, characterized in that the rice WRKY Genes include OsWRKY1 Genes and / or OsWRKY57 Genes; the stated OsWRKY1 The nucleotide sequence of the gene is shown in SEQ ID NO:1; OsWRKY57 The nucleotide sequence of the gene is shown in SEQ ID NO:2. This invention demonstrates through gene editing technology that... OsWRKY1 It is a positive regulator in the synthesis of anthocyanins in rice. OsWRKY57 It is a negative regulator of anthocyanin synthesis in rice. By knocking out... OsWRKY1 Genes can reduce the anthocyanin content in rice grains, and overexpression can reduce the anthocyanin content. OsWRKY1 Genes that increase anthocyanin content in rice grains; while gene knockout increases anthocyanin content in rice grains. OsWRKY57 Genes that increase anthocyanin content in rice grains, through overexpression. OsWRKY57 Genes can reduce the anthocyanin content of rice grains. The two rice WRKY family genes screened in this invention provide a theoretical basis for cultivating high-quality rice varieties with high anthocyanin content. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The anthocyanin content and expression levels of anthocyanin synthesis-related genes in 27 black rice varieties were compared. OsWRKY Correlation analysis of gene expression levels; Figure 2 This is a schematic diagram of the structure of the PCR amplification vector pGTR used to construct the knockout vector. Figure 3 This is a schematic diagram of the structure of the empty vector pCXUN-U6PU3T used to construct the knockout vector; Figure 4 for OsWRKY1 Schematic diagram of the structure of the gene knockout vector pCXUN-U6PU3T-WRKY1; Figure 5 This is a schematic diagram of the structure of the empty vector pCAMBIA1300nu used for gene overexpression. Figure 6 for OsWRKY1 Schematic diagram of the structure of the gene overexpression vector pCAMBIA1300nu-WRKY1; Figure 7 for OsWRKY1 Detection results of gene knockout material (KO-WRKY1) and overexpression material (OE-WRKY1); Where A is OsWRKY1 The mutant sequence of the gene knockout material; B is... OsWRKY1 The mature grain color of gene knockout materials and the mature grain color of overexpression materials, as well as OE-WRKY1 OsWRKY1 The results of the detection of relative expression levels; Figure 8 for OsWRKY1 Analysis of anthocyanin content in mature seeds of gene knockout material (KO-WRKY1) and overexpression material (OE-WRKY1); Where A represents the C3G content in mature kernels; and B represents the P3G content in mature kernels. Figure 9 for OsWRKY57 Gene knockout vector pCXUN-U6PU3T- OsWRKY57 A schematic diagram of the structure; Figure 10 for OsWRKY57 Schematic diagram of the structure of the gene overexpression vector pCAMBIA1300nu-WRKY57; Figure 11 for OsWRKY57 Detection results of gene knockout material (KO-WRKY57) and overexpression material (OE-WRKY57); Where A is OsWRKY57 The mutant sequence of the gene knockout material; B is... OsWRKY57 The mature kernel color and OsWRKY57The color of mature seeds of the gene overexpression material OE-WRKY57, and the content of OE-WRKY57... OsWRKY57 The results of the detection of relative expression levels; Figure 12 for OsWRKY57 Analysis of anthocyanin content in mature seeds of gene knockout material (KO-WRKY57) and overexpression material (OE-WRKY57); Where A represents the C3G content in mature kernels; and B represents the P3G content in mature kernels. Detailed Implementation

[0018] This invention provides rice WRKY The application of genes in regulating anthocyanin content in crops, specifically in rice. WRKY Genes include OsWRKY1 Genes and / or OsWRKY57 Genes; the stated OsWRKY1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:6; OsWRKY57 The amino acid sequence encoded by the gene is shown in SEQ ID NO:18.

[0019] As one implementation method, the OsWRKY1 The nucleotide sequence of the gene is shown in SEQ ID NO:1; OsWRKY1 The CDS sequence of the gene is shown in SEQ ID NO:5. As one embodiment, the... OsWRKY57 The nucleotide sequence of the gene is shown in SEQ ID NO:2; OsWRKY57 The CDS sequence of the gene is shown in SEQ ID NO:17.

[0020] As one implementation method, the regulation of crop anthocyanin content according to the present invention includes any one or more of the following aspects: 1) overexpression OsWRKY1 Genes that increase anthocyanin content in crops; 2) Knockout or inhibition OsWRKY1 Gene expression reduces anthocyanin content in crops; 3) Knockout or inhibition OsWRKY57 Gene expression increases anthocyanin content in crops; 4) Overexpression OsWRKY57 Gene expression reduces anthocyanin content in crops. As one embodiment, regulating crop anthocyanin content includes regulating anthocyanin content in crop grains. As one embodiment, the crop of this invention includes monocotyledonous plants. As one embodiment, the monocotyledonous plant includes rice.

[0021] This invention also provides a method for regulating rice WRKY Application of gene-expressing biomaterials in increasing anthocyanin content in crops, specifically rice. WRKY Genes include OsWRKY1Genes and / or OsWRKY57 Genes; the stated OsWRKY1 The gene encodes an amino acid sequence as shown in SEQ ID NO:6; OsWRKY57 The amino acid sequence encoded by the gene is shown in SEQ ID NO:18.

[0022] This invention also provides a method for regulating rice WRKY Biological materials for gene expression, said biological materials including any one or more of the following aspects: 1) overexpression of said gene... OsWRKY1 2) Contains the primer pairs of the gene; OsWRKY1 3) Gene recombinant vectors; OsWRKY1 Gene or 2) recombinant microorganisms of the recombinant vector; 4) knockout of the gene OsWRKY57 5) Targeting the gene's target sequence; OsWRKY57 Primer pairs for the target sequence of the gene; 6) Overexpression of the gene. OsWRKY57 7) Contains the primer pairs of the gene; OsWRKY57 8) Gene recombinant vectors; OsWRKY57 The recombinant microorganism of the gene or the recombinant vector described in 7); OsWRKY1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:6; OsWRKY57 The amino acid sequence encoded by the gene is shown in SEQ ID NO:18.

[0023] As one implementation, 1) the nucleotide sequences of the primer pair are shown in SEQ ID NO:3 and SEQ ID NO:4.

[0024] In one embodiment, 2) the recombinant vector includes an overexpression recombinant vector and a knockout recombinant vector. In another embodiment, the recombinant vector includes a backbone vector, which includes a plasmid vector. In another embodiment, the plasmid vector includes pCXUN-U6PU3T.

[0025] In one embodiment, 3) the recombinant microorganism includes overexpressed recombinant microorganisms and knockout recombinant microorganisms. In one embodiment, the recombinant microorganism includes basic microorganisms; in one embodiment, the basic microorganism includes bacteria. In one embodiment, the bacteria include Agrobacterium; in one embodiment, the Agrobacterium includes Agrobacterium strains. EHA105 .

[0026] As one implementation, the target sequence described in 4) is shown in SEQ ID NO:19.

[0027] As one implementation, the primer pair for the target sequence described in 5) is shown in SEQ ID NO:20 and SEQ ID NO:21.

[0028] As one implementation, the primer pair described in 6) is shown in SEQ ID NO:22 and SEQ ID NO:23.

[0029] In one embodiment, 7) the recombinant vector includes an overexpression recombinant vector and a knockout recombinant vector. In one embodiment, the recombinant vector includes a backbone vector, which includes a plasmid vector. In one embodiment, the plasmid vector includes pCXUN-U6PU3T-edit.

[0030] In one embodiment, 8) the recombinant microorganisms include overexpressed recombinant microorganisms and knockout recombinant microorganisms. In one embodiment, the recombinant microorganisms include basic microorganisms. In one embodiment, the basic microorganisms include bacteria. In one embodiment, the bacteria include Agrobacterium; in one embodiment, the Agrobacterium includes Agrobacterium strains. EHA105 .

[0031] This invention also provides a breeding method for increasing the anthocyanin content in crop grains, comprising one or both of the following steps: 1) overexpressing the anthocyanin in the crop. OsWRKY1 Genes to increase anthocyanin content in crop grains; OsWRKY1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:6; 2) Knockout or inhibition in crops OsWRKY57 Gene expression to increase anthocyanin content in crop grains; OsWRKY57 The amino acid sequence encoded by the gene is shown in SEQ ID NO:18.

[0032] This invention also provides a method for creating a biological model with low anthocyanin content, comprising the steps of any one or two of the following: 1) overexpressing the aforementioned in crops. OsWRKY57 Genes to reduce anthocyanin content in crops; OsWRKY57 The amino acid sequence encoded by the gene is shown in SEQ ID NO:18; 2) Knockout or inhibition in crops OsWRKY1 Gene expression to reduce anthocyanin content in crops; OsWRKY1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:6.

[0033] To further illustrate the present invention, the rice provided by the present invention will be described below with reference to the accompanying drawings and embodiments. WRKYThe genes and their applications in regulating anthocyanin content in crops are described in detail, but these should not be construed as limiting the scope of protection of this invention. In this study, the black rice variety “Lua_Chua_Chan” (Zheng J, et al. Determining factors, regulation system, and domestication of anthocyanin biosynthesis in rice leaves. New Phytol. 2019, 223: 705–721.) from 533 core rice germplasm resources was selected as the recipient rice variety for transformation. The knockout vector PGTR / pCXUN-U6PU3T and the overexpression vector pCAMBIA1300nu were both preserved in our laboratory.

[0034] Example 1 Rice WRKY Gene identification Twenty-seven black rice samples and forty-nine white rice samples collected in our laboratory were grouped into a population (Zhou Z, et al. RNA-seq reveals transcriptional differences in anthocyanin and vitamin biosynthetic pathways between black and white rice. Gene. 2022, 844:146845.). The white rice population included subgroups such as indica, japonica, aus, and intermediate types, while the black rice population consisted only of indica and japonica rice. Immature seed samples taken 10 days after flowering were analyzed by HPLC to determine the anthocyanin content, including two main anthocyanins: cyanidin-3-O-glucoside (C3G) and peony-3-O-glucoside (P3G). The determination method is referenced in "Zheng J, et al. Determining factors, regulation system, and domestication of anthocyanin biosynthesis in rice leaves. New Phytol. 2019, 223: 705–721."

[0035] Gene expression differential analysis was performed on the seed coat transcriptome of the population materials described above (screening criteria: |log2(Fold change)|>1, adjusted P-value). (Padj)<0.05), identifying a group of genes with differentially expressed levels in black and white rice seed coats. These differentially expressed genes, in addition to known transcription factors involved in anthocyanin synthesis regulation such as MYB, WDR, bHLH, and bZIP, also included genes from the WRKY transcription factor family. Subsequently, the differentially expressed genes were... WRKY Correlation analysis was performed on gene expression levels with anthocyanin synthesis-related genes and seed coat anthocyanin content (correlation and significance were calculated using the Pearson test, confidence interval: 0.95). The analyzed data were plotted using the corrplot package in R language. The results are shown below. Figure 1 As shown in Tables 1-1 and 1-2.

[0036] according to Figure 1 As shown in Tables 1-1 and 1-2, this embodiment preliminarily identified six rice pheromones that may be related to anthocyanin synthesis. WRKY Genes, that is OsWRKY1 , OsWRKY9 , OsWRKY12 , OsWRKY19 , OsWRKY23 , OsWRKY57 .in, OsWRKY1 (NCBI accession number: LOC_Os01g14440) showed the highest correlation with the expression levels of genes related to anthocyanin synthesis in black rice and the anthocyanin content in the seed coat. OsWRKY57 The expression levels of genes related to anthocyanin synthesis in black rice and the anthocyanin content in the seed coat showed a high correlation, therefore... OsWRKY1 and OsWRKY57 Further verification of gene function will be conducted.

[0037] in, OsWRKY1 The gene sequence is shown in SEQ ID NO:1: OsWRKY57 The gene sequence is shown in SEQ ID NO:2:

[0038] Table 1-1 Anthocyanin content, anthocyanin synthesis-related gene expression levels, and other parameters in 27 black rice varieties. OsWRKY Correlation coefficient of gene expression levels ( r )

[0039] Table 1-2 Anthocyanin content, anthocyanin synthesis-related gene expression levels, and other parameters in 27 black rice varieties. OsWRKY Correlation coefficient of gene expression levels ( r )

[0040] In Tables 1-1 and 1-2, Anth represents the anthocyanin content.

[0041] Example 2 OsWRKY1 CDS region isolation of genes Lua_Chua_Chan seeds were cultured into seedlings, and total RNA was extracted from Lua_Chua_Chan leaves using the Trizol method (Invitrogen). The extracted total RNA was treated with RNase-free DNase I (Invitrogen) and then reverse transcribed using M-MLV Reverse Transcriptase (Invitrogen). Oligo(dT) from the kit was added at 500 μg / μL. 15 100 μg of primer (Promega) was used to treat the RNA at 70°C for 10 min, and then the RNA was quickly placed on ice for 5 min to renature.

[0042] Add the following reagents to the processed RNA sample: 2 μL 5× first strand buffer, 0.5 μL 0.1 M DTT, 0.2 μL 10 mM dNTP, 1.3 μL DEPC H2O, and 0.25 μL M-MLV Reverse Transcriptase. Mix carefully and incubate at 37℃ for 1 h. After reverse transcription, inactivate the RNA at 75℃ for 5 min, add 10 μL DEPC water, and mix well to obtain total cDNA. according to OsWRKY1 Primers were designed based on the gene sequence information (NCBI accession number: LOC_Os01g14440) to amplify the CDS region of the Lua_Chua_Chan gene using total cDNA as a template. The primer sequences are shown below: OsWRKY1-CDS-F (SEQ ID NO:3):5'-ATGGAGGCCGACGACAGCGGC-3'; OsWRKY1-CDS-R (SEQ ID NO:4):5'-TTACTGCGTCTCGCTGTTGGTG-3'.

[0043] The PCR reaction system consisted of: 1 μL DNA template, 2.0 μL 10× buffer, 1.5 μL 2 mM dNTP, 0.2 μL each of 10 mM primers (OsWRKY1-CDS-F / R), 1 U rTaq polymerase, and ddH2O added to a final volume of 20 μL.

[0044] The PCR reaction program was as follows: 98℃ for 5 min; 98℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, 30-35 cycles; 72℃ for 10 min; 25℃ for 1 s.

[0045] The annealing temperature in the PCR reaction procedure varies depending on the primers used, generally ranging from 45℃ to 70℃. The extension time is set according to the size of the amplified product fragment; the theoretical amplification rate of rTaq is approximately 1 kb / min. The amplified products are detected by electrophoresis on a 1% agarose gel (containing the dye Goldview) and then sequenced.

[0046] The rice obtained by this invention OsWRKY1 The nucleotide sequence of the gene CDS is shown in SEQ ID NO:5, and the amino acid sequence it encodes is shown in SEQ ID NO:6.

[0047] OsWRKY1 protein SEQ ID NO:6:*.

[0048] Example 3 OsWRKY1 Construction and genetic transformation of gene knockout vectors and overexpression vectors A knockout vector was constructed using a multi-target CRISPR / Cas9 vector method for monocotyledonous plants. First, the rice described in Example 2 was used as an example. OsWRKY1 The CDS sequence of the gene was obtained using the online tool http: / / rice.hzau.edu.cn / cgi-bin / rice_rs3 / CRISPR_rice. OsWRKY1 One knockout target site with a 237 bp interval was selected from the full-length sequence. The knockout target site is shown below: Target1 (SEQ ID NO:7): 5'-GCGCAGTTCGCGCAACCGAGG-3'; Target2 (SEQ ID NO:8): 5'-ACGCAGTAAAAACGAGTTCGG-3'; Primers were designed targeting the above-mentioned target sites. The forward and reverse primers contain the 20 nt target sequence and a portion of the sequence from the intermediate vector, respectively. Specifically, the uppercase letters represent the target sequence on the genome, and the lowercase letters represent the portion of the sequence from the intermediate vector. The primer sequences are shown below: Cri-OsWRKY1-F (SEQ ID NO:9): 5'-ctgcgctgaggctcaggttGCGCAGTTCGCGCAACCGgttttagagctagaaatagc-3'; Cri-OsWRKY1-R (SEQ ID NO: 10): 5'-gctatttctagctctaaaacAACTCGTTTTTACTGCGTgccacggatcatctgcaca-3'.

[0049] Using the pGTR vector as a template, such as Figure 2 As shown, the PCR fragment was amplified using Cri-OsWRKY1-F and Cri-OsWRKY1-R as primers.

[0050] The PCR amplification system consisted of: 1 μL pGTR, 10 μL 2×PCR Buffer for KOD FX, 2 μL 2mM dNTPs, 0.2 μL each of Cri-OsWRKY1-F and R, 0.3 μL KOD (1 U / μL), and 6.3 μL ddH2O.

[0051] The PCR reaction program was as follows: 98℃, 3 min, 1 cycle; 98℃, 10 sec, 50℃, 20 sec, 72℃, 15 sec, 30 cycles; 72℃, 1 min, 25℃, 1 min, 1 cycle.

[0052] After confirming the PCR product size of 237 bp by agarose gel electrophoresis, the product was then excised and recovered using a DNA extraction kit (FastPure Gel DNA Extraction Mini Kit, Novizan).

[0053] use Kpn I. Enzyme digestion of the backbone vector pCXUN-U6PU3T, such as Figure 3 As shown, the enzyme digestion system is as follows: pCXUN-U6PU3T 2μL, FuniCut™ Kpn I 1 μL, 10×FuniCut™ Buffer 2 μL, ddH2O 15 μL, to perform the ligation reaction of the PCR fragment with the linearized vector backbone.

[0054] The ligation product was added to competent DH5α *E. coli* cells and transformed using the heat shock method. After recovery on a shaker at 37°C for 1 h, the cells were plated onto LB agar plates containing kanamycin and incubated at 37°C for 1 day. Four single colonies were randomly selected from the plates and placed into 2 mL sterile EP tubes (pre-filled with 800 μL of liquid LB medium containing kanamycin). The culture was shaken for 1 day, and plasmids were extracted and sequenced for verification. OsWRKY1 Knockout vector pCXUN-U6PU3T-WRKY1, the structure of pCXUN-U6PU3T-WRKY1 is as follows Figure 4 As shown.

[0055] The pCXUN-U6PU3T-WRKY1 plasmid was transformed into Agrobacterium strain. EHA105 The system consisted of 20 μL of Agrobacterium. EHA105 Competent cells + 1 μL plasmid, incubate on ice for 5 min, flash freeze in liquid nitrogen for 5 min, incubate in water at 37℃ for 5 min, then on ice for 5 min. Add 100 μL of antibiotic-free LB medium, incubate at 28℃ and 200 rpm for 2 h, then plate directly onto LA plates resistant to botulinum toxin, and incubate at 28℃ for two days. After two days, pick a single Agrobacterium colony from the plate and place it into a 2 mL sterile EP tube. Pre-fill the EP tube with 800 μL of liquid LB medium containing 50 μg / mL botulinum toxin, and incubate overnight on a shaker. The next day, send the bacterial culture for sequencing. After sequencing confirmation, the prepared Agrobacterium Cri-OsWRKY1 is ready for subsequent transformation experiments.

[0056] The reference for the Agrobacterium-mediated genetic transformation method is: Lin Yongjun, Chen Hao, Cao Yinglong, Wu Changyin, Wen Jing, Li Yafang, Hua Hongxia. Establishment of an efficient transgenic system for Mudanjiang No. 8 mediated by Agrobacterium, 2002, 28: 294-300. The specific steps include callus induction culture, callus subculture, Agrobacterium infection, co-culture, screening for hygromycin-resistant callus, followed by differentiation, rooting, hardening, and transplanting (the culture medium and its preparation method in this embodiment are described in patent number 2011100832269), to obtain T0 generation transgenic rice seedlings.

[0057] rice OsWRKY1 The steps for constructing the gene overexpression vector are as follows. Refer to Example 2. OsWRKY1 Design primers based on the cDNA sequence of the gene: OE-OsWRKY1-F (SEQ ID NO:11): 5'-aggtcgactctagaggatccATGGAGGCCGACGACAGCGGC-3'; OE-OsWRKY1-R (SEQ ID NO:12): 5'-gctggtcagagctcggtaccTTATTGCCCTCCGGCGTTTGG-3'.

[0058] The gene was cloned using cDNA from black rice (Lua_Chua_Chan) as a template and amplified using high-fidelity DNA polymerase. OsWRKY1 The CDS sequence, the nucleotide sequence of which is shown in SEQ ID NO:4, and the deduced amino acid sequence of which is shown in SEQ ID NO:5.

[0059] The PCR amplification system consisted of: 1 μL DNA, 10 μL 2×PCR Buffer for KOD FX, 2 μL 2mM dNTPs, 0.2 μL each of OE-OsWRKY1-F / R, 0.3 μL KOD (1 U / μL), and 6.3 μL ddH2O.

[0060] The PCR reaction program was as follows: 94℃, 2 min, 1 cycle; 98℃, 10 sec, 55℃, 30 sec, 72℃, 2 min, 30 cycles; 72℃, 1 min, 25℃, 1 min, 1 cycle.

[0061] After confirming the correct size of the PCR products by agarose gel electrophoresis, the products were then extracted using a DNA recovery kit (FastPureGel DNA Extraction Mini Kit, Novizan).

[0062] The structure of the overexpression vector pCAMBIA1300nu is as follows: Figure 5 As shown. By Figure 5 As can be seen, this embodiment uses Kpn I and BamH I. Double enzyme digestion was performed. After complete digestion was confirmed by agarose gel electrophoresis, the digested vector was purified using the sodium acetate method. The target gene and the linearized vector were ligated using homologous recombinase (ClonExpress II One Step Cloning Kit, Novizan). The ligation product was introduced into competent *E. coli* DH5α cells, and positive bacterial colonies were selected and sequenced on LB agar containing kanamycin. After successful alignment, the pCAMBIA1300nu-WRKY1 vector was successfully constructed. The structure of the pCAMBIA1300nu-WRKY1 vector is shown below. Figure 6 As shown, the above-mentioned plant expression vector was transferred into the rice variety Lua_Chua_Chan using the Agrobacterium-mediated transformation method to obtain T0 generation transgenic rice seedlings.

[0063] Example 4: Detection of positive transgenic plants After the transformed seedlings obtained in Example 2 were transferred into a greenhouse and allowed to grow to the four-leaf stage, their genomic DNA was extracted using the CTAB method. The specific steps are as follows: (1) Take a young leaf about 2 cm long and put it into a 2 mL centrifuge tube. Grind it with a grinder and add 800 μL of 1.5 × CTAB extraction buffer (the extraction buffer formula is: 15 g CTAB + 75 mL 1 M Tris-HCl (pH 8.0) + 30 mL 0.5 MEDTA + 61.4 g NaCl, and make up to 1000 mL with ddH2O). (2) Incubate in a 65℃ water bath for 30 min, inverting the tube every 5 min to mix. (3) In a fume hood, add 600 μL of chloroform to each sample tube and invert for 5-10 min. (4) Centrifuge at 8000 r / min for 10 min, and transfer 400 μL of supernatant to a new centrifuge tube. Add 600 μL of isopropanol and mix. (5) Centrifuge at 12000 r / min for 15 min. (6) Discard the liquid, invert the container onto absorbent paper to blot dry the surface liquid, and add 1 mL of 75% ethanol and mix well. (7) Centrifuge at 7500 r / min for 5 min. (8) Discard the liquid, invert the container onto absorbent paper to blot dry the surface liquid, blow dry the liquid on a clean bench, and add 100 μL of sterile ddH2O to dissolve the DNA.

[0064] Take 1 μL of the extracted genomic DNA as a template, and use online tools according to the knockout target site. https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / Design primers with the following sequences: Cas-WRKY1-F (SEQ ID NO:13):5'-GCAGTTCCAGGTGCCGCTGC-3'; Cas-WRKY1-R (SEQ ID NO:14):5'-CCTGGTTTGCAGCAGCAAA-3'.

[0065] PCR was used to detect the knockout plants. The PCR amplification system consisted of: 1 μL DNA template, 2 μL 10× buffer, 1.5 μL 2 mM dNTPs, 0.2 μL each of 10 mM primers (OsWRKY1-CDS-F / R), 1 μL rTaq polymerase, and ddH2O added to a final volume of 20 μL. The PCR reaction conditions were: 98℃ for 5 min; 98℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, for 30-35 cycles; 72℃ for 7 min; 25℃ for 1 s.

[0066] One independent transgene knockout was obtained through screening. OsWRKY1The family lineage, named KO-WRKY1, has the following sequencing results: Figure 7 As shown in Figure A, the knockout family exhibits a 241 bp deletion at the target sequence location of the target gene. This is because the knockout sequence cannot perfectly correspond to 237 bp, but is slightly longer than 237 bp. Furthermore, due to... Figure 7 As shown in the left image of B, compared with the wild-type control, the mature seed coat color of the KO-WRKY1 transgenic family changed from black to lighter, indicating a significant reduction in anthocyanin accumulation in the seed coat.

[0067] Will get OsWRKY1 Overexpression assays were performed using quantitative real-time polymerase chain reaction (qRT-PCR).

[0068] Total RNA extracted from rice was treated with RNase-free DNase I (Invitrogen) and then reverse transcribed using M-MLV Reverse Transcriptase (Invitrogen). 500 μg / μL Oligo(dT) was then added. 15 100 μg of primer (Promega) was added and treated at 70°C for 10 min, then immediately placed on ice for 5 min to renature the RNA. The following reagents were added to the treated RNA sample: 2 μL of 5× first strand buffer, 0.5 μL of 0.1 M DTT, 0.2 μL of 10 mM dNTP, 1.3 μL of DEPC H₂O, and 0.25 μL of M-MLV Reverse Transcriptase. The mixture was carefully mixed and incubated at 37°C for 1 h. The system that had completed reverse transcription was then inactivated at 75°C for 5 min, and 10 μL of DEPC water was added and mixed thoroughly.

[0069] The relative quantitative detection of gene expression levels involved in this experiment was achieved using qRT-PCR, and the expression levels of endogenous rice were used as the basis for detection. GAPDH The gene (NCBI accession number: LOC_Os04g40950) was used as an internal reference, and the relative expression level of the gene was measured using 2-1. -ΔΔCt The calculation method was used for evaluation, with three replicates per sample. The kits and qRT-PCR instruments used in the experiments were a Roche FastStart Universal SYBR Green Master (ROX) and an ABI ViiA7 Real-time PCR System. The qRT-PCR reaction mixture consisted of 3 μL cDNA template and 10 mM primers (qPCR- WRKY1Add 0.2 μL each of -F / R, 5 μL of 2 × Rox SYBRGreen I Mix, and bring the ddH2O to a final volume of 10 μL. qRT-PCR reaction conditions: 95℃ for 10 min; 95℃ for 10 s, 60℃ for 40 s, for 40 cycles.

[0070] The primer sequences are shown below: qPCR- WRKY1 -F (SEQ ID NO:15): 5'-AAGAGCCGGAGCAGCAACGA-3'; qPCR- WRKY1 -R (SEQ ID NO: 16): 5'-GGTCGGAGGCTGCATCATTT-3'.

[0071] This experiment screened and obtained three independent transgene overexpression sites. OsWRKY1 The families were named OE-WRKY1-1, OE-WRKY1-2, and OE-WRKY1-3, respectively. qRT-PCR analysis results showed that the above three OE-WRKY1 families... OsWRKY1 The relative expression level of the gene was increased by 8-10 times compared with the wild-type control (see results). Figure 7 (See Figure B on the right and Table 2). Furthermore, compared to the wild-type control, the mature seed coat color of the three OE-WRKY1 transgenic families was somewhat darker, indicating a possible increase in anthocyanin accumulation in the seed coat (results are shown in Figure B). Figure 7 (As shown in the left figure of B in the middle).

[0072] Table 2T0 generation WRKY1 Overexpression transgenic families and their control seeds OsWRKY1 relative expression level

[0073] Example 5: Anthocyanin content analysis in transgenic rice seeds Anthocyanins are water-soluble pigments; therefore, in this embodiment, the anthocyanin extraction solution was first prepared as follows: 105 mL extraction solution = 80 mL (chromatographic grade) pure methanol + 20 mL ddH2O + 5 mL 5 mol / L HCl solution. Furthermore, the mobile phase A for HPLC analysis consisted of ddH2O and (chromatographic grade) pure formic acid (volume ratio 200:1); the mobile phase B consisted of (chromatographic grade) pure methanol.

[0074] In this embodiment, the method for extracting anthocyanins is as follows: Fresh rice leaves and seed coat tissues are ground into powder using liquid nitrogen; mature rice seeds are ground into powder using a sample grinder. Approximately 0.1 g of leaf or seed coat tissue powder and 20 mg–30 mg of mature seed powder are weighed using an analytical balance and added to a 2 mL centrifuge tube; then 1 mL of extraction buffer is added to the sample, and the mixture is ultrasonically extracted for 1 h and then placed in a -20°C refrigerator for overnight extraction; after brief centrifugation, the supernatant is collected, filtered through a 0.22 μm filter membrane to remove impurities, and then injected into a 2 mL brown sample vial for analysis.

[0075] Mobile phase setup: Anthocyanin content was quantitatively determined using an HPLC-1260 (Agilent, USA) and a ZORBAX C18 SB-Aq 4.6×250 mm, 5 μm column; the mobile phase setup is shown in Table 3; the flow rate was 0.8 mL / min, the detection wavelength was 520 nm, the sample loading volume was 10 µL, and the injection was performed three times. The two most common anthocyanins in black rice, cyanidin-3-O-glucoside (C3G) and paeoniflorin-3-O-glucoside (P3G), were used as standards and were purchased from Miragen. The two standards were serially diluted to different concentrations: C3G was diluted to 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, and 3.125 μg / mL; P3G was diluted to 18.5 μg / mL, 9.25 μg / mL, 4.625 μg / mL, 2.312 μg / mL, 1.156 μg / mL, and 0.578 μg / mL. The content of the diluted standard was determined and a standard curve was plotted.

[0076] The results are as follows Figure 8 As shown in Table 4, the C3G content in the knockout material seeds was 0.55±0.04 mg / g; the P3G content was 0.07±0.01 mg / g, which were significantly lower than those in the wild-type control (C3G: 0.85±0.05 mg / g, P3G: 0.12±0.02 mg / g). The C3G content in the seeds of the three overexpression materials ranged from 1.35±0.25 mg / g to 1.43±0.12 mg / g; the P3G content ranged from 0.17±0.03 mg / g to 0.20±0.02 mg / g, which were significantly higher than those in the wild-type control (C3G: 0.85±0.05 mg / g, P3G: 0.12±0.02 mg / g).

[0077] Table 3 Mobile phase ratios for high performance liquid chromatography

[0078] Table 4. Anthocyanin content (mg / g) of seeds from the T0 generation WRKY1 transgenic family and its control.

[0079] Example 6 OsWRKY57 CDS region isolation of genes Based on the results selected in Example 1, this example uses the method described in Example 2 to... OsWRKY57 The difference in gene verification experiments lies in the primers used in this embodiment, which are based on... OsWRKY57 The primers were designed based on gene sequence information, and their specific sequences are shown below: OsWRKY57-CDS-F (SEQ ID NO:17):5'-ATGGCACACGGCGGCGGCGAGG-3'; OsWRKY57-CDS-R (SEQ ID NO: 18): 5'-AAATGTGCAAGTGGTTGCTTAA-3'.

[0080] The rice obtained in this embodiment OsWRKY57 The nucleotide sequence of the gene CDS is shown in SEQ ID NO:19, and the amino acid sequence it encodes is shown in SEQ ID NO:20.

[0081] OsWRKY57

[0082] OsWRKY57 protein (SEQ ID NO:20):MAHGGGEEEEERVLSHGDVVLLRCDLTILRGPHFLNDRIIAFYLAHLAADHHDDDLLLLPPSVPYLLSNLPDPASVAAVADPLRLASRRLVLLPVNDNPDVSHAEGGSH WTLLVLDNSNAVSGPRFVHHDSLPPTNLPSARRLAAVLRPLLPASAIPLIEGPTPRQTNGYDCGVFVLAVARAICNWWPTRARHSNSDSDWLEAVKREVNADSVKAMRTQLLQLIH TLIQNNTTTNQHSPSTQLPSHPSIASTSLPVTAQDFGTWLEDSGTHTAYDQKKADTGKGACWDNLTVSQSVRKPNVSAKNSLSYDGYSWRKYGQKQVKGSEFPRSYYKCTHPTCPV KRKVEMTPDGRIAEIVYNGEHNHPKPHPPRKPTLSTSVETLVATNDAGLENKLEGCDQAIGSDAVVEALRGGCHCLDGFRNGNEISDCKKRYAYAVIFIQNLLMFFCAKNVQVVA*.

[0083] Example 7 OsWRKY57 Construction and genetic transformation of gene knockout vectors and overexpression vectors This embodiment is constructed using the same method as in Embodiment 3. OsWRKY57 Gene knockout vectors, the difference is that, OsWRKY57 The gene knockout target is shown in SEQ ID NO:21.

[0084] Target3 (SEQ ID NO:21): 5'-GCTACAGCTGGAGGAAGTAT-3'; Primers were designed targeting the above targets. The forward and reverse primers contain the 20 nt target sequence and a portion of the sequence from the intermediate vector, respectively. The primer sequences are shown below: Cri-OsWRKY57-F (SEQ ID NO:22): 5'-GCTACAGCTGGAGGAAGTATgttttagagctagaaatagcaagtta-3'; Cri-OsWRKY57-R (SEQ ID NO:23): 5'-ATACTTCCTCCAGCTGTAGCAACCTGAGCCTCAGCGCAGC-3'.

[0085] The structure of the PCR amplification vector pGTR used in this embodiment for knockout vector construction is as follows: Figure 2 As shown, the PCR amplification method was the same as in Example 3. After confirming that the PCR product size of 1000 bp was correct by agarose gel electrophoresis, the product was excised and recovered using a DNA recovery kit (FastPure Gel DNA Extraction Mini Kit, Novizan).

[0086] use Kpn I enzyme digestion as Figure 3 The backbone vector pCXUN-U6PU3T-edit shown was obtained using the same enzyme digestion system and transformation method as in Example 3, and was verified by sequencing. OsWRKY57 The knockout vector pCXUN-U6PU3T-OsWRKY57 is described below. Figure 9 As shown. Following the same method described in Example 3, Agrobacterium pCXUN-U6PU3T-OsWRKY57 was prepared for subsequent transformation experiments.

[0087] rice OsWRKY57 The steps for constructing the gene overexpression vector are as follows. Refer to Example 6. OsWRKY57 Primers were designed based on the cDNA sequence of the gene. The primer sequences are shown below: OE-OsWRKY57-F (SEQ ID NO:24): 5'-CGAACGATAGCCGGTACCATGGCACACGGCGGCGGCGAG-3'; OE-OsWRKY57-R (SEQ ID NO:25): 5'-ATCTTTGTAATCGGATCCAGCAACCACTTGCACATTTTTTGGCAC-3'.

[0088] The gene was cloned using cDNA from black rice (Lua_Chua_Chan) as a template and amplified using high-fidelity DNA polymerase. OsWRKY57 The CDS sequence, its nucleotide sequence is shown in SEQ ID NO:19, and the deduced amino acid sequence is shown in SEQ ID NO:20. The PCR operation method is the same as in Example 3, except that OE-OsWRKY57-F / R is used as the primer. The CDS sequence is as follows: Figure 5 The overexpression vector pCAMBIA1300nu shown is used Kpn I and BamH I. Perform double enzyme digestion. After confirming complete digestion by agarose gel electrophoresis, purify the enzymes using the sodium acetate method. The enzyme digestion and purification methods are the same as in Example 3. Once the comparison is confirmed to be correct, the process is complete. Figure 10The overexpression vector pCAMBIA1300nu-OsWRKY57 was constructed as shown. The plant expression vector was transformed into the rice variety Lua_Chua_Chan using the Agrobacterium-mediated transformation method described above, resulting in T0 generation transgenic rice seedlings.

[0089] Example 8: Detection of positive transgenic plants After the T0 generation transgenic rice seedlings obtained in Example 7 were transferred into a greenhouse, their genomic DNA was extracted and primers were designed according to the method described in Example 4. The difference is that the designed primer sequences are as follows: KO-WRKY57SSR-F (SEQ ID NO:26): 5'-CTTGTAGTAGCTCCGTGG-3'; KO-WRKY57SSR-R (SEQ ID NO:27): 5'-ATGTCAGTGCGAAGAACA-3'.

[0090] The PCR method described in Example 4 was used to detect the knockout plants, and a total of 3 independent transgene knockouts were obtained. OsWRKY57 The families were named KO-WRKY57-1, KO-WRKY57-2, and KO-WRKY57-3, respectively. Sequencing results are as follows: Figure 11 As shown in Figure A, the three knockout families have deletions of 1 bp, 3 bp, and 13 bp at the target sequence position of the target gene, respectively.

[0091] In addition, the mature seeds of three KO-WRKY57 families, three OE-WRKY57 families, and WT showed similar appearances. Figure 11 As shown in the left image of section B, compared to the wild-type control, the mature seeds of the three KO-WRKY57 transgenic families have a darker seed coat color, indicating a significant increase in anthocyanin accumulation in the seed coat. The mature seeds of the three OE-WRKY57 transgenic families show almost no anthocyanin accumulation in the seed coat, indicating that anthocyanin accumulation in the seed coat is inhibited.

[0092] Will get WRKY57 Overexpression assays were performed using real-time quantitative polynucleotide chain reaction (qRT-PCR). The assay method was the same as in Example 4, except that the primers used for qRT-PCR were as follows: qpcr-wrky57-2F (SEQ ID NO:28): 5'-CATCTACCAGTCTGCCTGTCACA-3'; qPCR-wrky57-2R (SEQ ID NO:29): 5'-CAGCTGTAGCCATCGTAGGACA-3'.

[0093] This experiment screened and obtained three independent transgene overexpression sites. OsWRKY57 The family lineages are named OE-WRKY57-1, OE-WRKY57-2, and OE-WRKY57-3.

[0094] The results of qRT-PCR analysis of the OE-WRKY57 family are as follows: Figure 11 The right figure and Table 5 are shown in section B.

[0095] Table 5T1 generation WRKY57 Overexpression transgenic families and their WT OsWRKY57 relative expression level

[0096] Depend on Figure 11 As can be seen from the right figure of B and Table 5, among the above three OE-WRKY57 families... OsWRKY57 The relative expression level of the gene was increased by 5 to 13 times compared with the wild-type control.

[0097] Example 9: Anthocyanin content analysis in transgenic rice seeds This embodiment uses the three KO-WRKY57 transgenic families described in Example 8 for the experiment. The anthocyanin extraction solution and anthocyanin extraction operation are the same as in Example 5.

[0098] Mobile phase setup: Anthocyanin content was quantitatively determined using an HPLC-1260 (Agilent, USA) and a ZORBAX C18 SB-Aq 4.6×250 mm, 5 μm column; the mobile phase setup is shown in Table 3; the flow rate was 0.8 mL / min, the detection wavelength was 520 nm, the sample loading volume was 10 µL, and the injection was performed three times. The two most common anthocyanins in black rice, cyanidin-3-O-glucoside (C3G) and paeoniflorin-3-O-glucoside (P3G), were used as standards and were purchased from Miragen. The two standards were serially diluted to different concentrations. For C3G, the concentrations were determined by dilutions of 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL, 7.8125 μg / mL, 3.90625 μg / mL, 1.953125 μg / mL, 0.9765625 μg / mL, and 0.48828125 μg / mL. For P3G, the concentrations were determined by dilutions of 60 μg / mL, 30 μg / mL, 15 μg / mL, 7.5 μg / mL, 3.75 μg / mL, 1.875 μg / mL, 0.9375 μg / mL, 0.46875 μg / mL, 0.234375 μg / mL, and 0.117188 μg / mL, and a standard curve was plotted.

[0099] Analysis results as follows Figure 12 As shown in Tables 6 and 7, the C3G content in the seeds of the three knockout materials was 0.929±0.047 mg / g, 0.717±0.020 mg / g, and 0.677±0.016 mg / g, respectively; the P3G content was 0.087±0.016 mg / g, 0.069±0.011 mg / g, and 0.073±0.021 mg / g, respectively. These values ​​were significantly higher than those of the wild-type control (C3G: 0.478±0.024 mg / g, P3G: 0.079±0.005 mg / g). Figure 12 (Table 6). The C3G content in the seeds of the three overexpression materials was 0.009±0.001 mg / g, 0.015±0.002 mg / g, and 0.013±0.002 mg / g, respectively; the P3G content was not detected in any of them, and was significantly lower than that of the wild-type control (C3G: 0.478±0.024 mg / g, P3G: 0.079±0.005 mg / g). Figure 12 (Table 7).

[0100] Table 6. Anthocyanin content (mg / g) of seeds from the T1 generation KO-WRKY57 transgenic family and its control.

[0101] Table 7. Anthocyanin content (mg / g) of seeds from the T1 generation OE-WRKY57 transgenic family and its control.

[0102] In summary, this invention has demonstrated through gene editing technology that... OsWRKY1 It is a positive regulator in the synthesis of anthocyanins in rice. OsWRKY57 It is a negative regulator of anthocyanin synthesis in rice. By knocking out... OsWRKY1 Genes can reduce the anthocyanin content in rice grains, and overexpression can reduce the anthocyanin content. OsWRKY1 Genes that increase anthocyanin content in rice grains; while gene knockout increases anthocyanin content in rice grains. OsWRKY57 Genes that increase anthocyanin content in rice grains, through overexpression. OsWRKY57 Genes can reduce the anthocyanin content of rice grains. The two rice WRKY family genes screened in this invention provide a theoretical basis for cultivating high-quality rice varieties with high anthocyanin content.

[0103] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments without creative effort, as shown in these embodiments, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Rice WRKY The application of genes in regulating crop anthocyanin content is characterized by, The rice WRKY Genes are OsWRKY1 Gene; The OsWRKY1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:6; The regulation of crop anthocyanin content includes regulating the anthocyanin content in crop grains; The crop in question is rice; The regulation of crop anthocyanin content includes any one or more of the following aspects: 1) Overexpression OsWRKY1 Genes increase anthocyanin content in crops; 2) Knockout or suppression OsWRKY1 Gene expression reduces anthocyanin content in crops.

2. Regulating rice WRKY The application of gene-expressing biomaterials in increasing anthocyanin content in rice is characterized by, The rice WRKY Genes are OsWRKY1 Gene; The OsWRKY1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:6; The biomaterial includes any one or more of the following aspects: 1) Contains the above OsWRKY1 Gene recombination vectors; 2) Contains the above OsWRKY1 Recombinant microorganisms of the gene or the recombinant vector described in 1); Increasing the anthocyanin content in rice includes increasing the anthocyanin content in rice grains.

3. A breeding method for increasing the anthocyanin content in rice grains, characterized by the following steps: include: Overexpression in rice OsWRKY1 Genes to increase the anthocyanin content in rice grains; The OsWRKY1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:

6.

4. A method for creating a biological model with low anthocyanin content, characterized in that the steps include... include: Knockout or inhibition in rice OsWRKY1 Gene expression to reduce anthocyanin content in rice; The OsWRKY1 The amino acid sequence encoded by the gene is shown in SEQ ID NO:6.

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